Method for treating tumors with a toxin

ABSTRACT

A method for treating a tumor is provided, which comprises the steps of preliminarily immunizing a patient in need of antitumor treatment with a toxin or toxin surrogate vaccine in an amount which is effective to generate an immune response to the toxin in the patient, thereby providing systemic protection from the toxin to the patient, and subsequently administering the toxin to the patient in an amount which is effective to kill tumor cells. The toxin may be any suitable toxin, for example ricin, abrin, gelonin or diphtheria.

This is a divisional application of Ser. No. 08/486,200, filed Jun. 7,1995 now U.S. Pat. No. 5,667,786.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The instant invention is a novel method for treating tumors with atoxin. More specifically, the present method involves preliminarilyimmunizing a patient with a toxin vaccine containing a cytotoxiccancerostatic toxin or toxin surrogate to generate an immune response tothe toxin in the patient and to thereby provide systemic protection fromthe toxin in the patient, and then administering the cytotoxiccancerostatic toxin to the patient to directly treat the tumor.

2. Description of Related Art

Various methods for treating tumors using toxins have been known and arepresently under investigation. The most common of these methods isimmunotoxin therapy, which involves administering to a cancer patientusually by systemic infusion a toxin linked to a peptide ligand. Theligand portion of the immunotoxin is usually an antibody or hormone. Theligand of the immunotoxin directs the molecule to the surface of thetumor cell, where the toxin enters the cell to kill the cell. Mosttoxins work by inactivating the cell ribosomes, thereby stopping proteinsynthesis in the cell. See, e.g., Oncology, "Immunotoxin Therapy ofCancer", May 1993.

While effective in some cases, several problems have been recognizedwith immunotoxin therapy, making it clear that immunotoxin therapy isnot, at least so far, the "silver bullet" for cancer treatment that itwas once hoped to be.

One problem with immunotoxin therapy is that the large size of theimmunotoxin molecules and the poor vasculature of most tumors leads to ahigh intravascular drug concentration but a low drug concentration inthe tumor interstitia. Consequently, toxicities to accessible normaltissues is high, while efficacy is poor. For example, immunotoxins havebeen found to react with neural and hepatic tissue antigens and produceserious, and sometimes fatal, toxicities.

Another problem with immunotoxins is that they are significantlyimmunogenic, generating humoral immune responses to the molecule. Theresulting immune complexes of antibody-immunotoxin reduce the effectivelevel of immunotoxin systemically by, for example, inhibiting theinternalization of the immunotoxin into the tumor cell.

A second known method for treating tumors with a toxin involves theintravenous treatment of a tumor using a sub-lethal dose of the toxin.This treatment, about which there is only one report known to thepresent inventors, demonstrated an antitumor effect of the toxin inhuman clinical trials, but the patients had adverse side effects at highdoses. In addition, since toxins are notably immunogenic, the patientsmounted an immune response to the toxin which eventually abrogated anyeffect of the treatment.

Most notably, the above report on the clinical experiment concluded thatintravenous treatment of a tumor with a toxin suffered from a problem ofhaving a limited duration of effective therapy. The report concludedthat the therapy becomes ineffective when antibody titers to the toxinin the patient reach a concentration sufficient to counteract or abolishthe antitumor effect of the toxin. See Godal et al., Int J. Cancer,"Antibody Formation Against the Cytotoxic Proteins Abrin and Ricin inHumans and Mice", Vol. 32, pp 515-521 (1983).

SUMMARY OF THE INVENTION

It is an object of the invention to provide an improved method forintravenous treatment of a tumor with a toxin.

More specifically, it is an object of the invention to minimize the sideeffects produced in patients from the toxin to be administered.

It is a further object of the invention to extend the duration ofeffective toxin therapy, curtailed in present methods by the immuneresponse mounted to the toxin which is considered to eventually abrogateany effect of the treatment.

It is a still further object of the invention to avoid the problem ofimmunotoxin therapy in having a low drug concentration at the tumorsite.

The present invention has been achieved by the discovery by the presentinventors that the afore-mentioned problems can be solved bypreliminarily immunizing the patient with the toxin or toxin surrogateas a vaccine. As with conventional vaccines, the toxin vaccine provokesan immune response which is sufficient to provide systemic protection tothe patient. It is then possible to directly treat the tumor with thetoxin, for example by direct intratumoral injection of lethal doses ofthe toxin into the tumor. The systemic protection of the patient againstthe toxin protects the normal tissues of the patient from any toxinwhich might escape from the tumor.

Thus, in contrast to the intravenous treatment method mentioned abovewhich considered the development of the patient's immune response to thetoxin to be a disadvantage, the present invention turns the immuneresponse into an asset for the treatment of tumors.

To the knowledge of the present inventors, the antitumor treatmentmethod of the present invention characterized by a pre-immunization ofthe tumor patient with a toxin vaccine to develop a systemic immunity tothe toxin, before administering the toxin to directly kill the tumorcells, is entirely novel and unsuggested in the prior art.

The present invention will now be explained in more detail hereinbelow.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

The present method is suitable for treating tumors in humans, non-humanmammals (monkey, mouse, etc.), pets (dog, cat, hamster, etc.), livestock(cow, horse, pig, sheep, goat, etc), and zoo animals (elephant, giraffe,etc.).

The present method is suitable for treating both solid tumors (liver,pancreas, breast, brain, lung, head, neck, etc.) and non-solid tumors(leukemia, B-cell lymphoma, etc.). Treatment of solid tumors isparticularly preferred.

The toxins which are useful in the method of the present invention aregenerally any known cytotoxic cancerostatic protein toxins. Ricin,abrin, gelonin, exotoxin A and diphtheria are preferred.

Systemic protection from the toxin in the patient is achieved by anactive immunization of the patient with a sub-lethal dose of the wholetoxin, or a toxin surrogate. The term toxin surrogate means a sub-unitor portion of the toxin capable of stimulating the immune response, or atoxoid derivative of the toxin.

It is known in the art that an active protective immune response can beachieved by sub-lethal doses of a toxin (Hewetson et al., Vaccine,"Protection of mice from inhaled ricin by vaccination with ricin or bypassive treatment with heterologous antibody", Vol. 11, Issue 7, 1993;Godal et al., Int. J. Cancer, cited above), a toxoid (Rippy et al., Soc.Tox. Path., 1991) or sub-units of a toxin (Lemley et al., Programme andAbstracts, Third Asia-Pacific Congress on Animal, Plant and MicrobialToxins, Jun. 27-Jul. 1, 1993).

The whole toxin or sub-units of the toxin can be obtained fromcommercial sources, such as from Inland Laboratories, Austin, Tex., anddiluted in a pharmaceutically acceptable carrier. Sub-units of the toxincan be prepared by known methods from the whole toxin.

A toxoid preparation of the toxin can be prepared by conventionalmethods, such as by stirring with formaldehyde (10% formaldehyde whichis 3.7% formalin in phosphate buffered saline) for 28 days at 4° C.followed by extensive dialysis against phosphate buffered saline.

Formalin inactivation of whole ricin can be achieved by incubating withformaldehyde for 3 days followed by stationary phase, surface to airevaporation to precipitate the vaccine is described on page 418 ofLemley et al., Hybridoma, "Identification and Characterization of aMonoclonal Antibody that Neutralizes Ricin Toxicity In Vitro and InVivo", Vol. 13, No. 5, 1994.

Licensed vaccines of some toxins, for example diphtheria, arecommercially available. Methods for preparing vaccines and generatingantibodies are also known in the art. For example, methods forgenerating antibodies to diphtheria in humans and mice are alsodescribed in Godal et al., Int. J. Cancer, cited above.

To achieve systemic protection, the amount of the toxin or toxinsurrogate to be administered the patient is in the range from 1.0-1,000μg/dose. A 5.0-10.0 μg/dose is preferred. These dosage ranges apply togenerally all known toxins and routes of administration. The dosageranges also generally apply to any potential patient regardless ofweight. Thus, the amount of toxin to be administered a mouse can beabout 5.0 μg/dose, while the amount of toxin to administer to a humancan be about 10.0 μg/dose.

One skilled in the art can readily determine the optimal dosage of toxinor toxin surrogate to administer to a patient to achieve systemicprotection using known methods. For example, the optimal dosage may bedetermined by conducting a clinical trial administering escalatingdosages of the toxin beginning at the lower dosage range to determine adosage which affords the desired level of systemic protection. Systemicprotection can be evaluated standard methods, such as ELISA(titer >300), cell assay or by neutralizing the toxin with sera andinjecting into naive mice.

The toxin may be optionally administered with a conventional adjuvant toenhance the immune response. Only alum is presently approved for use inhumans as an adjuvant. For animal uses, a plurality of adjuvants areapproved and may be used.

The toxin or toxin surrogate vaccine is administered by any conventionalroute for administering vaccines. Intramuscular, subcutaneous, oral,topical, mucosal and aerosol are preferred.

The minimum effective amount of toxin to achieve systemic protection isa single dose of about 10.0 μg of formalin treated toxin/alum absorbed.

In the most preferred embodiment, the patient is immunized with thetoxin vaccine, including an adjuvant, in a single intramuscularinjection, or 2-3 i.m. injections, of 5.0-10.0 μg. In the case ofmultiple dosages, the dosages are preferably given two weeks apart. Inthe case of a single dosage, the first toxin treatment can operate as abooster.

Direct treatment of the tumor with the toxin is begun preferably abouttwo weeks after immunization. The toxin must be the same toxin used inthe immunization step.

Whole toxin is preferably administered in most cases, not a toxinsurrogate, although an active derivative or portion of the whole toxinmight be used if the molecule is cytotoxic and cancerostatic.

The toxin can be administered in any acceptable pharmaceutical medium.The toxin is usually administered in a sterile saline solution. Thetoxin may optionally contain a known protein stabilizing agent which isnot immunogenic, such as human albumin in the case of treating humanpatients.

The toxin may be administered to the patient through any conventionalroute. An intratumoral delivery of the toxin directly into the tumor ispreferred, achieved by infusion or catheter. In the case of theintravenous injection of the toxin, the systemic protection of thepatient is expected to protect the normal tissues from the toxin whilekilling the tumor cells having a faster growth rate rendering the tumorcells more susceptible to the toxin.

The toxin is administered in as many dosages as the patient can tolerateand as necessary to achieve an antitumor effect. Preferably one or twodoses of toxin are administered.

To achieve an antitumor effect, the amount of the toxin to beadministered the patient is in the range from 100 μg-1.0 mg per dose. Adose of 5.0-10.0 μg/dose is preferred. These dosage ranges apply togenerally all known toxins and routes of administration. The dosageranges also generally apply to any patient regardless of weight.

In the most preferred embodiment, the toxin is administered byintratumoral infusion or catheter in an amount of 10 to 100 times thelethal dose for the patient.

One skilled in the art can readily determine the optimal dosage of toxinto administer to a patient to achieve an antitumor effect using knownmethods, for example those methods used in treatment methods using knowntoxins discussed above, and those methods using other cytotoxicantitumor agents such as chemotherapeutic agents and ethanol. Theoptimal dosage may be determined by conducting a clinical trial usingescalating dosages of the toxin beginning at the lower dosage range todetermine a dosage of toxin which the patient can tolerate whileachieving an antitumor effect.

The method of the present invention also has the advantage of beinginexpensive, at least from the standpoint of the vaccine and toxin used.The cost of the vaccines is estimated to be in the range of $1.00-$5.00per dose. The cost of the therapeutic toxin in a dose of 100 μg-1.0 mgis estimated to be $20-$25/mg.

Hence, the pharmaceuticals used in the method of the present inventionprocedure are very inexpensive, making the therapy quite feasible forzoo animals, pets or livestock, as well as humans.

The present invention also includes a toxin or toxin surrogate vaccine,as described above, and a kit containing a vial or unit containing thetoxin or toxin surrogate vaccine together with a vial or unit containingthe toxin treatment.

We claim:
 1. A kit for treating a tumor, which comprises a first unitcontaining a toxin or a toxin surrogate vaccine in an amount which iseffective to generate an immune response to the toxin in a patient,thereby providing systemic protection from the toxin, and a second unitcontaining the toxin in an amount which is effective to kill tumorcells, said vaccine comprising ricin toxoid and adjuvant, and saidsecond unit comprising a tumor-killing effective amount of ricin insterile saline.
 2. A kit for carrying out a method of treating tumors,which comprises a first unit containing an adjuvant, and a toxin ortoxin surrogate vaccine in an amount that generates an immune responseto the toxin in a patient, thereby providing systemic protection fromthe toxin, and a second unit containing the corresponding toxin in theamount which is effective to kill tumor cells, said vaccine selectedfrom the group consisting of a ricin toxoid and a ricin-a-chain, andsaid second unit further containing sterile saline.
 3. The kit accordingto claim 2, which contains a unit containing a vaccine of ricin toxoid,an adjuvant, and a unit containing a tumor-killing effective amount ofricin in sterile saline.
 4. The kit according to claim 2, which containsa unit containing a vaccine of ricin a-chain, an adjuvant, and a unitcontaining a tumor-killing effective amount of the ricin in sterilesaline.